Cell culture conditions

The cell lines used in this study included LLC, A549, HFL1, Human Umbilical Vein Endothelial Cell (HUVEC), MH-S, and RAW264.7. These cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, in a 5% CO2 environment at 37 °C.

Animals

C57BL/6 female mice were procured from Guangdong Medical Laboratory Animal Center. All procedural animal methodologies followed the guidelines set by the Guangzhou Medical University Care and Use of Lab Animals, and the experiments received approval from the School of Guangzhou Medical University’s Animal Ethics Committee.

Preparation of CN-Pt

Preparation of the nitrogen-carbon material included the direct carbonization pyrolysis of chitosan. First, 1 g chitosan powder was placed in a quartz boat and positioned in the plasma enhanced chemical vapor deposition (PECVD) system. Then, all the air was removed from the internal tube of the PECVD system to create a vacuum before inert Argon (Ar2) gas was continuously introduced into the tube at a flow rate of 50 ml/min. Then ramp up the temperature was conducted at a rate of 10 °C/min until it reached 600 °C and a growth time of 4 h and a stop time for insulation was set. Finally, the nitrogen-carbon material was collected from the quartz boat and transferred to a reagent bottle. Then it was stored in a super clean glove box.

Preparation of the CN-600 two-dimensional nitrogen-carbon material. We added 100 mg nitrogen-carbon material powder to ultra-pure water. Using an ultrasonic crusher with a power of 200 W and a switch cycle of 2/2 s, material delamination was conducted for 8 h, after which the undelaminated large nitrogen-carbon material was separated to obtain CN-600 of different sizes. Then, cascade centrifugation was performed on the water dispersion solution to isolate that which contained large material blocks. This solution was then divided equally into two 50 ml centrifuge tubes and centrifuge at 2000 rpm for 10 min. The supernatant of the solution contained the large-sized CN-600 without block bodies. Sequential centrifugation of the supernatant at 4000, 8000, and 12,000 rpm for 10 min each, with the supernatant collected after each centrifugation used for the next round of centrifugation. The precipitate produced from each step included CN-600 nanosheets with gradually decreasing lateral dimensions.

To prepare the CN-600-PEG to improve the stability and biocompatibility of the CN-600 water dispersion, we performed a coating modification with DSPE-PEG-NH2. First, 20 mg CN-600 powder obtained by centrifugation at 8000 rpm was added to 10 mL ultrapure water. Then, a certain amount of DSPE-PEG-NH2 was added with a feed ratio of 1:5 (mCN-600:mDSPE-PEG-NH2 = 1:5). Next, the CN-600 and DSPE-PEG-NH2 mixture was placed on a magnetic stirrer at a constant temperature and stirred with a magnetic stir bar for 12 h. The mixture solution was centrifuged at 12,000 rpm for 10 min and the supernatant containing the dispersed DSPE-PEG-NH2 was collected while retaining the precipitate. The obtained precipitate was dried in a vacuum drying oven to obtain CN-600-PEG.

To prepare the metal precursor, 1 g hexahydrated chloroplatinic acid (H2Cl6Pt·xH2O) was added to a mixed solution of 25 mL ultrapure water and hydrochloric acid (ultrapure water: HCl = 24:1 (v/v) with 1 mol/L HCl). The mixture was stirred at constant temperature for 12 h using a magnetic stirrer to obtain a uniformly dispersed metal (Pt) precursor solution (H2Cl6Pt aqueous solution).

To synthesize CN-600-PEG-Pt, 20 mg CN-600 powder obtained by centrifugation at 8000 rpm was added into 10 mL ultrapure water. A certain amount of uniformly dispersed H2Cl6Pt aqueous solution was added to achieve a mass ratio of metal precursor to CN-600 of 2%. After the solution was prepared, it stirred at a constant temperature for 12 h using a magnetic stirrer. The mixture solution was centrifuged to remove the unloaded discrete platinum particles. The mixture solution was centrifuged at 12,000 rpm for 10 min and the supernatant was collected while retaining the precipitate. The precipitate was dried using a vacuum drying oven to obtain CN-600-PEG-Pt.

Transmission electron microscopy

The prepared CN-600-PEG and CN-600-PEG-Pt were dispersed in pure water. Then, 20 µL of the sample solution was dropped onto a carbon-coated copper grid. After complete drying, the morphology of CN-600-PEG and CN-600-PEG-Pt was observed using TEM.

The particle size and zeta potential

Solutions of CN-600-PEG and CN-600-PEG-Pt were prepared at a concentration of 0.5 mg/mL for use in a laser nanoparticle size analyzer to measure the particle size and zeta potential at a temperature of 25 °C. The measurements were recorded from three parallel experiments for each sample.

X-ray photoelectron spectroscopy

Ten milligrams of CN-600-PEG and CN-600-PEG-Pt were evenly spread on the sample test bench and the composition of the material was characterized by x-ray photoelectron spectroscopy (XPS), with the test elements being C, N, and Pt.

Preparation of SFMA

The degummed SF was obtained by immersing 10 g cocoon in 1 L 0.05 M NA2CO3 solution and boiling it at 100 °C for 30 min, followed by several washes with distilled water. The degummed SF was then dried in an oven for 36 h. To obtain SFMA, 10 g degummed SF was dissolved in 9.3 M LiBr solution at 60 °C for 1 h. The mixture was stirred using a magnetic stirrer and then slowly dripped into 6 mL glycidyl methacrylic acid. After 8 h, the solution was passed through a filter cloth to remove the salt and dialyzed for 7 d using a dialysis bag and distilled water. The obtained SFMA solution was frozen at -80 °C for 12 h and then freeze-dried for 36 h to obtain spongy SFMA.

Preparation of GelMA

Gelatin was dissolved in 250 mL distilled water at 60 °C before 12 mL methacrylic anhydride was added and the reaction was incubated for 8 h at room temperature, followed by dialysis with distilled water for 3–5 days (retention molecular weight: 3500). The dialyzed solution was decolorized with activated carbon and centrifuged at 8000 rpm before it was passed through a neutral filter paper for lyophilization at -80 °C to obtain GelMA.

Gel permeation chromatography

A 5 mg aliquot of SFMA and GelMA each were dissolved in pure water and then filtered through a 0.22-µm strainer before being injected it into the GPC system to test the molecular weight fraction of the sample.

Preparation of hydrogels

In the process of formulating composite hydrogels, the GelMA concentration was consistently maintained at 6% (w/v) in PBS, which was combined with varying quantities of SFMA (2% (w/v), 4% (w/v), and 6% (w/v)) in PBS and mixed into the GelMA solution at a volume ratio of 1:1. Subsequently, 0.1% (w/v) of the photo-initiator, LAP, was added and the solution was irradiated with 405 nm UV light for 10 s to obtain the GelMA/2% SFMA, GelMA/4% SFMA, and GelMA/6% SFMA hydrogels. Similarly, using this preparation method, a GelMA/4% SFMA solution was obtained and then add the desired concentration of Gemcitabine solution and a final concentration of 300 µg/mL CN-Pt solution with 0.1% (w/v) LAP and 405 nm UV light irradiation was used to obtain Hydrogel/CN-Pt and Hydrogel/CN-Pt/Gem.

1H NMR test

Deuterium was used instead of heavy water as the solvent to dissolve the SF, SFMA, Gel, and GelMA samples. The samples were added to a clean MRI tube after they were completely dissolved and clarified, and then their chemical structure was characterized using 1H NMR (Inova-500 M Varian company, America) at room temperature. The atlas was analyzed using MestReNova software.

Scanning electron microscopy

For SEM, 400 µL of the hydrogels (GelMA, GelMA/2% SFMA, GelMA/4% SFMA, GelMA/6% SFMA, and GelMA/4%SFMA/CN-Pt) were stored in a refrigerator at -80 °C for one night, dried, and surface-coated with gold for 30 s. The surface morphologies of the hydrogels were observed using SEM (S-3400, Hitachi, Japan) with a 5-kV electron beam.

Swelling rate test

The swelling rate test of the hydrogels was observed using a gravimetric method. In this test, 400 µL of each hydrogel (GelMA, GelMA/2% SFMA, GelMA/4% SFMA, GelMA/6% SFMA, and GelMA/4% SFMA/CN-Pt) were placed in a water bath at 37 °C for 15 min and then demolded. After measuring the dry weight (Wdry) of the hydrogel, it was placed in PBS (pH 7.4) at 37 °C. The swollen hydrogels were removed from the PBS and weighed (Wswollen) at predetermined time points after removing excess water using a filter paper. The water absorption ratio (Q) of the hydrogels was calculated as follows: Q = (Wswollen- Wdry)/Wdry × 100%.

Rheological test

The rheological behavior of the hydrogels was determined using a 25-mm-diameter stainless steel parallel plate rotary head. The samples (GelMA, GelMA/2% SFMA, GelMA/4% SFMA, GelMA/6% SFMA, and GelMA/4% SFMA/CN-Pt) were scanned by the rheometer (Kinexus, UK Malvern) at room temperature from 0.1 to 10 rad/s to determine the linear viscoelasticity range of the hydrogels and measure the storage modulus (elastic modulus, G′) and loss modulus (viscous modulus, G″). The curves of G′ and G″ were recorded.

Compression test

Compression tests were evaluated using a universal testing machine (Dynamic universal testing machine ELF3200; Dr. America). In these tests, 600 µL of the hydrogels GelMA, GelMA/2% SFMA, GelMA/4% SFMA, GelMA/6% SFMA, and GelMA/4% SFMA/CN-Pt) were placed under the gel probe, which squeezed the gel until it broke. The force required to break the hydrogel was recorded and defined as the strength of the hydrogel.

Preparation of the GEM standard curve

Two milligrams of precisely weighed GEM was added to a 10 mL bottle and dissolved in methanol to obtain the GEM stock solution, which was then diluted with methanol to concentrations of 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg/mL, and 100 µg/mL. GEM detection was performed using an ultraviolet spectrophotometer at 175 nm. The optical density (OD) value was used as the ordinate, sample concentration was used as the abscissa, and a standard curve was prepared.

In vitro GEM release rates

For the in vitro release studies, 600 µL of each hydrogel were placed in 2 mL PBS (pH = 7.4) solution and incubated at 37 °C. A sample of the supernatant was collected at each time point and replaced with the same volume of fresh PBS. The collected PBS supernatants were tested for GEM using a photometer to calculate the cumulative release rate.

Influence of hydrogel concentration on the photothermal effects of the materials

A cylindrical hydrogel was prepared and inserted into a thermocouple thermometer at room temperature. The hydrogel was irradiated with 808-nm NIR (power, 1.5 W/cm2) for 5 min and the temperature was recorded at 10 s intervals. Using time point was used as the abscissa and temperature values as the ordinate. The photothermal effects of the hydrogels with different concentrations of CN-Pt were compared in vitro. A blank hydrogel was used as the control.

Influence of laser power on the photothermal effects of the materials

The liquid surface was irradiated with NIR at different powers (0.5, 1, 1.5, and 2 W/cm2) at 808 nm for 5 min. A thermocouple thermometer was inserted into the hydrogel and the temperature was recorded every 10 s at room temperature. A blank hydrogel was used as the control.

Photothermal stability of the materials

The hydrogel was irradiated with 808-nm NIR (power, 1.5 W/cm2) for 5 min and the irradiation was stopped to reduce the temperature to the initial temperature. The experiment was repeated five times. A thermocouple thermometer was inserted into the hydrogel and the temperature was recorded at 10 s intervals at room temperature.

Cytotoxicity assay

The cytotoxicity of the hydrogel system was evaluated with the CCK-8 assay. LLC, A549, HFL1, and HUVECs cells were cultured in 96-well plates (5 × 103 cells/well) for 12 h before different hydrogels were introduced to the cells. After incubation for 24, 48, or 72 h, the medium was removed and CCK-8 solution diluted in fresh medium was added. After 2 h of incubation, the absorbance was measured at 450 nm using a microplate reader (K3 Plus, BIODL) and cell viability was calculated. To further verify the CCK-8 results, a live/dead staining assay was carried out using calcein and propidium iodide as described earlier, and the cells were visualized using a fluorescence inverted microscope (Zeiss, Germany).

Apoptosis assay

Cell apoptosis was detected using the Annexin V 633 apoptosis detection kit. The cells were gathered and stained with Annexin V/PI for 30 min at room temperature. The samples were then analyzed using flow cytometry (BD, United States).

Western blotting analysis

Cells were subjected to lysis by RIPA lysis buffer containing protease and phosphatase inhibitors. Protein extracts were resolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature and then incubated with primary antibodies against GAPDH (Proteintech; 60004-1-Ig, 1:10000), MLKL (Abclonal; A13451), p-MLKL (Abclonal; AP0949, 1:1000; Boster, P00535, 1:1000), RIPK1 (Abclonal; A7414, 1:1000), p-RIPK1 (Abclonal; AP1115, 1:1000), RIPK3 (Abclonal; A5431, 1:1000), and p-RIPK3 (Abcam; ab209384, 1:2000; Abcam, ab222320, 1:1000) overnight at 4 °C. The membranes were then washed with PBS with Tween-20 (PBST) and incubated with secondary antibodies slowly. Using enhanced chemiluminescence (ECL) (Biosharp), the signals were detected using a Tanon infrared imaging system (Guangzhou EWELL Bio-Technology Co., LTD., China).

DAMPs

A549 cells were treated with different treatment for 24 h. The cells were subsequently rinsed three times with ice-cold PBS and then incubated with anti-CRT antibodies (Proteintech, CL650-27298, 1:200) at 4 °C for 30 min before conducting flow cytometry (BD, United States). The supernatants of the treated A549 cells were also for HMGB1 detection using an enzyme-linked immunosorbent assay (ELISA) kit (MM-13713H1; MEIMIAN). Detection of extracellular ATP release was conducted using an ATP assay kit (S0026; Beyotime).

qRT–PCR

Total RNA was extracted using AG RNAex Pro Reagent AG21102 [Accurate Biotechnology (Human) Co., Ltd]. RNA was reverse transcribed into cDNA using the Evo M-MLV RT Premix for qPCR AG11706 (Accurate Biotechnology (Human) Co., Ltd). The cDNA was processed in an Applied SYBR Sequence Detection System (LightCycler 480 II, Roche) using a real-time quantitative PCR (SYBR Green, Invitrogen). The primer sequences were as follows:

M-GAPDH-F: CCACCCCAGCAAGGAGAC.

M-GAPDH-R: GAAATTGTGAGGGAGATGCT.

M-IL-12β-F: GGAGACCCTGCCCATTGAACT.

M-IL-12β-R: CAACGTTGCATCCTAGGATCG.

M-IL-23-F: TGGAGCAACTTCACACCTCC.

M-IL-23-R: GGGCAGCTATGGCCAAAAAG.

M-CD-86-F: ATGGACCCCAGATGCACCAT.

M-CD-86-R: TAGGTTTCGGGTGACCTTGC.

Phagocytosis assay

RAW264.7 LLC cells were treated with Dio and Dil, respectively, for 15 min. The RAW264.7 cells were incubated with the same number of LLC cells in the different treatment groups for 4 h at 37 °C. Phagocytosis was stopped by washing with 4 °C PBS and centrifugation at 1000 rpm/min. The cells were detected using flow cytometry (BD, United States).

The animal model for anti-tumor efficacy

To investigate the therapeutic potential of the hydrogel system, 1 × 106 Luc-LLC cells were injected into the backs with C57BL/6 mice (6–8 weeks of age). After 12 d, mice were randomly divided into six groups (n = 6), anesthetized, and approximately 99% of the tumors were surgically removed with sterilized instruments to leave approximately 1% residual tumor to mimic residual micro-tumors post-surgery. Immediately after surgery, various hydrogel treatments, including PBS, hydrogel, hydrogel/CN-Pt, hydrogel/CN-Pt + NIR, hydrogel/Gem, and hydrogel/CN-Pt/Gem + NIR, were applied and followed by the addition of 0.1% LAP and 405 nm UV light exposure for 10 s. The surgical tumor site was then closed by suturing. The tumor size was gauged and calculated using the formula: width2 × length × 0.5. The tumors were also evaluated with bioluminescence imaging monitoring. At a dose of 10 µL/g, D-luciferin (40901ES03, YEASEN) in DPBS (15 mg/mL) were intraperitoneally injected into each mouse. The mice were imaged using an IVIS imaging system for 3 min. Animals displaying signs of ill health or bearing tumors exceeding a size of 1.5 cm3 were euthanized.

Flow cytometry

The treated mice from each group were euthanized on day 14. The tumor tissues were dissected into small pieces and digested in 1 mg/mL collagenase IV and 20 µg/mL DNase I in 1640 complete medium at 37 °C for 60 min to obtain cell suspensions. Then, the cells were filtered through a 70 μm nylon cell strainer and used for flow cytometry analysis. The cells were blocked with CD16/CD32 (dilution of 1: 500) to reduce nonspecific antibody binding. For LIVE + CD45 + CD3 + CD4 + and LIVE + CD45 + CD3 + CD8 + T cell analysis, cells were stained with LIVE-APC-H7 (dilution of 1:3000), anti-CD45-PerCP-Cy5.5 (dilution of 1:300), anti-CD3-APC (dilution of 1:300), anti-CD4-BV510 (dilution of 1:300), and anti-CD8-FITC (dilution of 1:300). For LIVE + CD45 + CD11b + F4/80 + CD86 + and LIVE + CD45 + CD11b + F4/80 + CD206 + macrophage analysis, cells were stained with LIVE-APC-H7 (dilution of 1:3000), anti-CD45-PerCP-Cy5.5 (dilution of 1:300), anti-CD11b-FITC (dilution of 1:300), anti-F4/80-BV421 (dilution of 1:300), anti-CD86-PE-CY7 (dilution of 1:300), and anti-CD206-APC (dilution of 1:300). The cells were stained for 30 min at 4 °C and then washed with PBS. After centrifugation and resuspension, the cells were used for flow cytometry (BD, United States).

In vitro antibacterial experiments

The antibacterial performance of the hydrogels was evaluated using gram-positive S. aureus and gram-negative E. coli. The concentration of the bacterial suspension was adjusted to 1 × 106 CFU/mL and then 200 µL hydrogel sample were incubated with 100 µL bacterial suspensions. The samples in the GelMA/4% SFMA/CN-Pt group were exposed to NIR laser for 5 min. After treatment, 1.7 mL Luria-Bertani (LB) medium was added to the samples and the cells were cultured at 37 °C for 4 h. An aliquot of 100 µL of the diluted bacterial suspension was used to inoculate LB agar plates, which were cultured for 24 h at 37 °C before the number of culturable colonies was counted. The formula AR (%) = (Nc - Ns)/Nc × 100% was used to calculate the antibacterial rate (AR), where Nc is the average bacterial colony number of the control sample and Ns is the average bacterial colony number of the hydrogel sample.

In vivo antibacterial experiments

The rats were treated with 3% sodium pentobarbital sodium (45–60 mg/kg) before the hair around the surgical site was removed with an animal razor and the exposed skin was disinfected with a iodophor. The surgical instruments were sterilized using an autoclaved steam cooker. The skin on the back of the animals was removed using tissue shears. Four infection model wounds with circular skin defects were created on both sides of the back of each rat. The wound diameter was 12 mm and the distance between each wound was approximately 2 cm. The wound was infected with a mixture of 40 µL S. aureus (1 × 108 CFU / mL) and E. coli (1 × 108 CFU/mL). The medium was added 24 h after infection and the rat was returned to its corresponding cage alone. The change in wound size was recorded every 3 d for 14 d. At predetermined time points, wounds were excised from the mice for the production of paraffin sections, and HE and MT staining of the wound tissues were also performed.

Statistical analysis

The survival curves were analysed by using the log-rank (Mantel–Cox) test. Differences among multiple groups were evaluated using one-way ANOVA and t-test was used for two-group comparisons. Significant differences are indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.